The background information is believed, at the time of the filing of this patent application, to adequately provide background information for this patent application. However, the background information may not be completely applicable to the claims as originally filed in this patent application, as amended during prosecution of this patent application, and as ultimately allowed in any patent issuing from this patent application. Therefore, any statements made relating to the background information are not intended to limit the claims in any manner and should not be interpreted as limiting the claims in any manner.
Glycosylation of proteins is an important aspect of protein therapeutics. For example, many Food and Drug Administration (FDA) approved protein therapeutics, and those in clinical trials, are glycoproteins. A majority of FDA approved protein therapeutics may be glycosylated. Glycoproteins are proteins that have oligosaccharide chains or glycans attached to one or more sites on the polypeptide backbone. Glycosylation is the enzymatic linking of sugar molecules to produce oligosaccharides or glycans covalently attached to the protein. This process may result in significant heterogeneity in the composition and structure of attached glycans. This pattern of glycosylation may be crucial in biopharmaceutical development and manufacturing since it may influence binding, clearance, immunogenicity, and mechanism of action of the protein therapeutic.
Therefore, the glycan profile of the protein may need to be carefully monitored to ensure compliance to quality control standards. Typically, the determination of glycan profiles may be achieved by enzymatic cleavage of the glycans from the protein followed by chromatographic or mass spectrometric characterization. Although these methods may provide characterization of glycan structure, they may be time consuming, require expert interpretation, and may not be easily used for monitoring glycan profile changes during a production run in a manufacturing facility. For example, current methods for glycan characterization or methods of characterizing and/or identifying glycoproteins may be time consuming, on the order of 24 hrs or greater, and/or labor intensive. A less time consuming screening method for glycan composition may aid the industry by allowing near real time glycosylation monitoring during upstream processing, i.e., in a bioreactor, and also for bioprocess development.
Monoclonal antibodies (mAbs) are a particularly important therapeutic class because they are a fast growing class of protein therapeutic. All mAbs are glycosylated and the structure and composition of these sugar structures may be critical to their efficacy and safety. Currently, nearly all licensed therapeutic mAbs have been of the IgG (immunoglobulin G) class. The oligosaccharides or glycans of mAbs may be attached to two specific amino acid residues in the stem region of the antibody. In the IgG class, glycans (or oligosaccharides) may be covalently attached at asparagine 297 of each heavy chain in the Fc region of the mAb. The glycan component may affect the solubility, stability, immunogenicity and effector function of mAbs. It therefore may be essential to characterize and control the glycosylation of therapeutic mAbs.
The production of therapeutic mAbs with a consistent glycosylation pattern may currently present a considerable challenge to the biopharmaceutical industry. As a result, product release may require extensive characterization of oligosaccharide or glycan composition to ensure product quality and consistency. For example, chromatography or mass spectrometry are typically used to analyze the glycan content of mAbs. These methods of analysis of glycans may be time-consuming, may involve enzymatic cleavage of the glycans prior to analysis, and may require expensive instrumentation and highly-trained personnel.
There is a need for a method of characterizing glycans attached to glycoproteins that may overcome at least some of the deficiencies of the prior art.